Plants are potentially a low cost and contamination safe factory for the production of recombinant pharmaceutical proteins. Most of the recombinant proteins produced in plants are indistinguishable from their mammalian counterparts, as far as the amino acid sequence, conformation and biological activity. Furthermore, mammalian glycoproteins are efficiently glycosylated when they are expressed in transgenic plants. However, plants produce molecules with N-glycans that differ from those found on animal glycoproteins (Lerouge et al., 1998). This may limit the use of plant-made pharmaceuticals since the presence of plant-specific glyco-epitopes on these proteins may elicit immune responses in humans (Bardor et al., 2003) as well as the absence of mammalian-type epitopes, such as sialylated sequences, may induce their fast clearance from the blood stream. As a consequence, controlling the N-glycosylation of plant-made pharmaceuticals is a prerequisite for their use in human therapy.
In planta remodelling strategies have recently emerged to obtain plant-derived antibodies with human compatible carbohydrate profiles. Some strategies involved the retention of the plantibodies in the endoplasmic reticulum (Ko et al., 2003; Sriraman et al., 2004, Triguero et al., 2005), others involved the transformation of plants with mammalian glycosyltransferases. For example, plant N-glycosylation can be partially humanised by transformation of plant with a human β(1,4)-galactosyltransferase (Palacpac et al., 1999; Bakker et al., 2001). Expression of a murine antibody in a transformed plant resulted in the production of a plant-derived antibody harbouring a galactosylation profile similar to the one observed in the corresponding murine IgG (Bakker et al., 2001).
Mammalian IgGs bear bi-antennary N-glycans on the conserved site of N-glycosylation located in the Fc domain. These oligosaccharides are weakly sialylated, and the absence of terminal Neu5Ac does not interfere with the antibody function and stability. In contrast, most other circulatory glycoproteins have sialylated di-, tri or tetra antennary N-glycans. The presence of terminal sialic acids on these glycans is required for numerous biological functions, the first one being the control of the half-life of the protein in the circulatory system. In the absence of terminal sialic acids, glycoproteins are detected by hepatic asialoglycoprotein receptors and cleared from the serum, rendering these proteins biologically short-lived and ineffective (Kelm and Schauer, 1997). Therefore, non-sialylated plant-made pharmaceuticals may be rapidly eliminated from the blood stream when injected to a human, for example, a tobacco-derived Epo was biologically active in vitro but non functional in vivo because of its removal from the circulation before it reached erythropoietic tissues (Matsumoto et al., 1995).
Remodeling of N-glycans linked to plantibodies into human-like N-glycans has been already partially achieved in plants by expression of a human β(1,4)-galactosyltransferase (Palacpac et al., 1999; Bakker et al., 2001), a transferase that uses the endogenous UDP-Gal as co-substrate. A mammalian sialyltransferase has also been introduced in plants and demonstrated to be functional and correctly targeted to the Golgi apparatus (Wee et al., 1998). However, no sialylation of endogenous oligosaccharides was observed. The occurrence of sialic acids as well as the sialylation machinery in plants is still a matter of debate. However, Neu5Ac, the major sialic acid present in humans, as well as its precursor N-acetylmannosamine (D-ManNAc) do not appear to be synthesised in plants in detectable amounts (Séveno et al., 2004). As a consequence, the glyco-engineering of plant N-glycans into sialylated oligosaccharides requires the co-expression of exogenous enzymes able to catalyse the synthesis, the activation and the transfer in the Golgi apparatus of Neu5Ac.
In mammals and bacteria, anabolism and catabolism of Neu5Ac occurs through different pathways (Angata and Varki, 2002). Two main classes of enzymes are required to form Neu5Ac. N-acetylneuraminate lyases (Neu5Ac lyase) is involved in the catabolism of sialic acids by catalysing the cleavage of Neu5Ac into N-acetylmannosamine (D-ManNAc) and pyruvate in a reversible reaction. At high concentrations of D-ManNAc and pyruvate, the equilibrium can be shifted to the synthesis of Neu5Ac. Coupled to a glucosamine 2-epimerase activity, Neu5Ac lyase from E. coli was used for the large-scale production of Neu5Ac from D-GlcNAc (Maru et al., 1998). Alternatively, Neu5Ac synthases, such as NeuB, catalyze the condensation of ManNAc onto phosphoenol pyruvate (PEP) and are directly involved in the biosynthesis of sialic acids (reviewed in Tanner, 2005).